Project Summary: As mammalian cells prepare to divide, the abscission checkpoint checks for residual mitotic errors and delays abscission until the checkpoint is satisfied. Cells then complete cytokinesis and the two daughter cells are irreversibly separated. Recent work has shown that loss of the abscission checkpoint leads to accumulation of DNA damage and correlates with a predisposition to several different types of human cancer, indicating the checkpoint plays an important protective role. Although we lack a complete understanding of checkpoint signaling, it is already clear that the Endosomal Sorting Complexes Required for Transport (ESCRTs) are a key regulatory target of the checkpoint. During cytokinesis, ESCRT complexes are recruited to and function in the midbody between the dividing cells, where they constrict the membrane and mediate daughter cell separation. However, these ESCRT activities must be negatively regulated to prevent abscission until the checkpoint is satisfied. We have recently identified the protease Calpain-7 as a novel and essential checkpoint component. In preliminary studies, we have characterized how the Calpain-7 MIT domain binds the ESCRT-III protein IST1, determined the structure of the relevant Calpain-7 MIT-IST1 complex, identified point mutations that inhibit this interaction, shown that IST1 recruits Calpain-7 to the midbody, and demonstrated that Calpain-7 catalytic activity and ESCRT-III binding are required to maintain the abscission checkpoint. We now propose to gain a mechanistic understanding of how Calpain-7 maintains the checkpoint by identifying substrates of Calpain-7 proteolysis and characterizing their functions (Aim 1) and by performing biochemical and structural analyses of Calpain-7 regulation (Aim 2). Experiments in Aim 1 will utilize an unbiased, proximity-based TurboID/mass spectrometry approach to identify candidate Calpain-7 substrates. These candidates will then be validated by testing for Calpain-7 processing in vitro and characterizing their roles in abscission checkpoint maintenance in cultured cells. Experiments in Aim 2 will provide structural and biochemical insights into Calpain-7 regulation, including the mechanism of autoinhibition and the role of protein oligomerization in IST1 binding and midbody recruitment. Together, these studies will provide important new mechanistic insights into the regulatory networks and midbody assemblies that arrest cell division in response to mitotic errors.